Section 5: Chapter 17 Epidemiology

Erysipelothrix, Lactobacilllus, and Similar Organisms Erysipelothrix spp:

- Seen in vertebrate and invertebrate animals
(mammals, birds, fish, sheep, rabbits, cattle,
General Characteristics and turkey)
 Catalase-negative - Transmitted through direct contact or ingestion
 Non-soreforming of water/meat
 Gram positive - Assoc. with animals and contracted by humans
through animal exposure
Erysipelothrix rhusiopathiae – the only human
pathogen (in the genus) Arcanobacterium spp:

- Have serovars based on peptidoglycan - Normal inhabitants of mucosal membranes of

structure. cattle, sheep, dogs, cats, and pigs
֍ Serovars 1 and 2: most common in human Trueperella bernadiae
infx
- Skin abscesses
T.pyogenes
Arcanobacterium spp:
→ mucous membranes of cattle, sheep, and pigs
- Irregular, gram positive rods → Assoc. with animals and contracted by humans
- CAMP (+) through animal exposure
Arcanobacterium haemolyticum: the only one
recovered in clinical specimens
Pathogenesis and Spectrum of Disease
G. vaginalis and Lactobacillus – natural inhabitants of
Trueperella spp (T. pyogene and T. bernardiae) the human vagina.
- CAMP test (-) » Vaginal infections with G. vaginalis are often
found in association with a variety of mixed
anaerobic flora.
Gardnerella vaginalis – gram (+), thin layer of » Extravaginal infx: uncommon, assoc. with
peptidoglycan layer postpartum endometritis, septic abortion, and
- Gram variable rod or coccobacilli cesarean birth
- Part of normal human microbiota Lactobacillus spp: imp. for maintaining the proper pH
balance in vaginal secretions.
Lactobacillus spp: - Metabolize glucose to lactic acid: acidic
vaginal pH  not conducive to the growth of
- May be beneficial to human but can cause pathogenic bacteria
infx to immunocompromised patients - Frequently associated with dental carries
- L. acidophilus - Enters the bloodstream during chewing,
- L. casei brushing teeth, and dental procedures
- L. fermentum - Bacteremia and endocarditis
- L. gasseri
- L. plantarum Weisella confusa
- L. rhamnosus - Lactobacillus-like organism
- L. ultunensis - Recovered in blood cultures from patients with
- Normal human microbiota clinical symptoms of endocarditis
- Vancomycin resistant
Weisella confusa – was known as L. confuses Erysipelothrix
- Assoc with individuals who are fish handlers,
farmers, slaughterhouse workers, food
preparation workers, and veterinarians
- puncture wound or skin abrasion
3 categories of human disease
1) localized skin lesions/ cellulitis (erysipeloid)
2) diffuse cutaneous infection with systemic
symptoms
3) bacteremia
Arcanobacterium spp. and Trueperella spp:
- animal pathogens
- assoc. with pathogens pharyngitis,
septicemia, tissue abscesses, and ulcers in
immunocompromised patients
Acarnobacterium haemolyticus – assoc. with
pharyngitis in human infections with mild to severe
symptoms
T. bernardiae: recovered from the blood, abscesses,
UT, joints, the eyes, and wounds
- implicated as the cause of necrotizing fasciitis
T.pyogenes: infx in patients from rural environments
- identified in abscesses, wounds, and blood
infections
Laboratory Diagnosis
Specimen Collection and Transport
No special considerations are required for specimen
collection and transport
Erysipelothrix: skin lesions
- collected by biopsy of the full thickness of skin
at the leading edge of the discolored area
Specimen Processing
No special considerations are required for processing
of the organisms
Direct Detection Methods
Gram staining of Arcanobacterium spp: delicate,
curved, gram-positive rods with pointed ends and
occasional rudimentary branching
- may stain unevenly after 48 hours of growth
on solid media and show coccal forms
Lactobacillus
- highly pleomorphic
- occurring long chaining rocs in coccobacilli
and spiral forms
T. bernardiae
» short
» gram positive rods without branching
E. rhusiopathiae:
 both short rods and long filaments
 2 colonial types:
o Rough colonies that contain slender,
filamentous, gram positive with
tendency to over-decolorize and
appear gram negative
o Smooth colonies that contains small,
slender rods
 May be mistaken for a polymicrobial infection
Gardnerella
◊ Small, pleomorphic, gram variable or gram-
negative coccobacilli and short rods
◊ Wet mount staining and Gram Staining
o Key tests for diagnosing bacterial
vaginosis by G. vaginalis
◊ Clue cells: large, squamous epithelial cells
with numerous attached small rods
◊ Gram-stain of the discharge: attached
organisms to be gram-variable coccobacilli
◊ Bacterial vaginosis: clue cells are present,
large numbers of other gram positive rods
(i.e., lactobacilli), normal vaginal microbiota
◊ BD Affirm vaginal DNA probe (VDP): used
from direct detection from genital specimen
◊ Special vials containing transport reagents:
used to stabilize the organism’s nucleic acids
before testing
Cultivation
Media of Choice
- Grown on 5% sheep blood and CAP
- Do not grow in Mac Conkey agar
- Grow on CAN agar
o CNA: nutritional base that may include
5% growth of gram-negative
organisms
- Colistin and nalidixic acid: prevent the over-
growth of gram-negative organisms
- All genera except Gardnerella spp grow in
commercially available blood culture broths
- Gardnerella organisms are inhibited by SPS
o Used as an anticoagulant in most
commercial blood culture
o SPS free medium: supplemented with
gelation should be used when G.
vaginalis sepsis
 - Isolated from female genital tract specimen:
Human Blood Bilayer Tween agar (HBT)
o CAN agar with amphotericin B added
to prevent the growth of yeasts and
filamentous fungi
o Human blood is layered over the top to
enhance the beta-hemolytic pattern of
G.vaginalis
Incubation Conditions and Duration
Detectable growth
- On 5% S-BA and CA
- CNA
- HBT
o Incubated at 35°C in 5% to 10% CO2
within 48 hours of inoculation
E. rhusiopathiae: produce H2S
Colonial Appearance
G.vaginalis – produces small, gray, opaque colonies
surrounded by surrounded by diffuse zone of beta-
hemolysis on HBT agar
Approach to identification
- Actinomyces
- Bifidobacterium
- Propionibacterium spp.
o Considered with anaerobic bacteria
o 5% to 10% CO2
o Catalase negative
o Gram positive
o Non-spore forming rods
- The HNID panel (Haemophilus-Neisseria
identification panel)
o Fastidious gram-negative bacterial
identifications
- Rapid identification: used for isolates from
extragenital sources (e.g., blood)
Matrix-Assisted Laser Desorption Ionization
Time-of-Flight Mass Spectrometry (MALDI-TOF
MS)
- Identification of E. rhusiopathiea
Comments Regarding Specific Organisms
- Presumptive identification of G. vaginalis
o Sufficient for genital isolates
o Based typical appearance on Gram
stain
o Beta-hemolysis on HBT
o Oxidase and Catalase (-)
- Corynebacterium lipophiloflavum: bacterium
isolated from females with bacterial vaginosis
 Catalase positive and
resembles C. urealyticum
- C. lipophiloflavum: producing a yellow
pigment, weak urease activity, and slow acid
production from glucose
- Arcanobacterium spp: beta-hemolytic
o A. haemolyticum & Trueperella
pyogenes (formerly A.pyogenes)
 Differentiated based on
liquefaction of gelatin
 T.pyogenes: positive
 A. haemolyticum: negative
→ Beta-hemolytic on
human blood agar
 Trueperella bernardiae (A.
bernardiae) =: nonhemolytic
Erysipelothrix spp.
- Only catalase-negative
- Gram-positive
- Non-spore forming rod
- Produces hydrogen sulfide (H2S)
- Inoculated into Triple Sugar Iron (TSI)
Bacillus spp
- Blacken the butt of TSI
- Catalase positive
- Produces spores
- Important to identify and report isolation of this
organism in clinical samples
- Intrinsic resistance to vancomycin has been
identified
Lactobacillus spp:
- Identified based on colony and Gram stain
morphologies
- Catalase negative
- Formation of rods in chain rather than cocci in
Thioglycollate broth is helpful
- Gram stain of growth just outside the zone of
inhibition surrounding the 10-U penicillin disk
placed on blood agar plate
- Inoculated with a lawn of the organism should
show long bacilli rather than coccoid rather
than coccoid
Antimicrobial Susceptibility Testing and Therapy
Lactobacillus spp: can be resistant to various
antimicrobial agents.

Susceptibility testing
- G. vaginalis: not recommended
- Typically treated with metronidazole
- Systemic infection: ampicillin or amoxicillin

Prevention
-ubiquitous in nature
Part of the normal human microbiome commonly
encountered without deleterious effect
No recommended vaccination or prophylaxis
Section 6: Chapter 18 ֍ Strict aerobic
֍ Branched filaments that extend along the agar
Norcardia, Streptomyces, Rhodococcus, and Similar o Substrate hyphae: surface
Organisms o Aerial hyphae: into the air
֍ Inc. age= fragments change into pleomorphic
Introduction
rods or coccoid elements
Actinomyces: large and diverse group of gram- ֍ Meso-diaminopimelic acid (DAP)
positive bacilli ֍ Arabinose and galactose (cell wall sugars)
a) N. asteroids
- Elongate b) N. brevicatena/ N. paucivorans
- Form brancing c) N. cyriacigeorgica
- Filamentous (hyphae) d) N. nova
Clinical relevant aerobic actinomyces genera: exhibit e) N. farcinica
branchin or partial acid-fastness f) N. brasiliensis
g) N. otitidiscavarium
Corynebacterium spp: do not show branching h) N. pseudobrasiliensis
filaments or partial acid-fastness i) N. transvalensis
Mycobacterium: spp: do not exhbit branching and are
strongly acid fast
Rhodococcus, Dietzia, Gordonia, and
2 groups of aerobic actinomycetes Tsukamurella spp
1. Cell walls with mycolic acid & partially acid-  Gram positive
fast  Aerobic
2. Cell walls do not contain mycolic acid – non-  Catalase (+)
acid fast  Partially acid-fasr
Genemic sequencing: used to delineate species  Branching
within this diverse phylogenetic group  Filamentous bacteria
 Fragment into rods and cocci
Rhodococcus – dismissed as diptheroids
General Characteristics
R. equi: separate new genus = Prescotella
- Partially acid-fast aerobic actinomyces
Nocardia & Rhodococcus: family Norcardiaceae
Non-Acid Fast Aerobic Actinomycetes:
Gordonia & Tsukamurella: families Gordoniaceae &
Tsukamurellaceae Streptomycetes, Actinomadura, Dermatophilus,
Nocardiopsis, and the Thermophilic
Actinomadura Actinomycetes
 52 species and subsp  Gram positive
 Cell wall: contain madurose  Branching filaments
o Shared characteristic with genus  Do not contain mycolic acids = non-acid fast
Dermatophilus  Heterogeneous
Thermophilic Acticomycetes
Partially Acid-Fast Aerobic Actinomycetes o Thermoactinomyces
Nocardia spp. o Saccharomonospora
o Saccharopolyspora
֍ Gram-positive
֍ Beaded app
֍ Variably acid-fast
֍ Catalase (+)
Epidemiology and Pathogenesis Non-Acid Fast Aerobic Actinomycetes:
PARTIALLY ACID-FAST AEROBIC Streptomycetes, Actinomadura, Dermatophilus,
ACTINOMYCETES Nocardiopsis, and the Thermophilic
Actinomycetes
Nocardia spp.
 Normal inhabitants of soil and water
 Responsible for the decomposition of plant
material
 Ubiquitous
 Isolation: colonization of the skin and URT
 Infections: Traumatic or inhalation inoculation
 Hematogenous spread after pulmonary foci = dse
in different body sites
o BRAIN – most prominent secondary site
of infx.
 N. asteriodes: the most common human
pathogen isolate
 N. asteroids sense stricto: rarely pathogenic
Virulence:
a) Growth at the time of infection
b) Resistance to intracellular killing
c) Tropism for neuronal tissue
d) Ability to inhibit phagosome-lysosome fusion
e) Production of large amounts of catalase
f) Hemolysins

Rhodococcus, Dietza, Gordonia, Tsukamurella

spp
 Isolated from environmental sources such as soil,
farm animals, fresh and salt water
 Infection on respiratory inhalation of organisms
 ROT: unknown
 Gordonia: isolated from catheter-related and
other medical device-associated infx. Spectrum of Disease
 Gordonia spp and Tsukamurella: opp. Infx in
Particially Acid-Fast Aerobic Actinomycetes
humas
Nocardia spp
Rhodococcus equi
 Can spead hematogenously
a. Assoc with human dse (immunocompromised
 Disseminated infx: lesions in brain and skin
= HIV)
o Involve CNS
b. Facultative intracellular organism
a. Can persist and replicate within 3 types of skin infections (immunocompetent)
macrophages
c. Virulence antigens A and B (VapA, VapB0 1) Mycetoma: chronic, localized, painless, subQ infx
d. Transmission: inhalation from exposure to 2) Lymphocutaneous infections
infected animal feces 3) Skin abscesses or cellulitis
In Immunocompromised
☼ Invasive pulmonary infection
☼ Disseminated infection Acute hypersensitivity pneumonitis
At risk: ◊ malaise
◊ chills
 Systemic immunosuppression
◊ sweats
 Transplant recipients
◊ loss of appetite
 Impaired pulmonary immune defense
◊ chest tightness
 IV drug abusers
◊ cough
SSX ◊ fever w/in 4-6hrs of exposure

 Fever Chronic hypersensitivity pneumonitis

 Night sweats
 progressively worsen with subsequent dev’p of
 Weight loss
irreversible lung fibrosis
 Productive cough
Pulmonary infx Compli:
Laboratory Diagnosis
 Pleural effusion
 Empyema Specimen Collection, Transport, and Processing
 Mediastinitis
 Soft tissue infection  No special reqs
 Material from draining sinus tracts: excellent spx
for direct examination and culture
Rhodococcus, Dietza, Gordonia, Tsukamurella Direct Detection Methods
spp
→ Gram staining: most important in diagnosis of
֍ Opportunistic pathogens aerobic actinomycetes
Occur in immunocompromised px → Aerobic actinomycete:
o Gram (+)
o Branching/ partially branching beaded
Non-Acid Fast Aerobic Actinomycetes: filaments
→ Modified acid-fast stain (1% sulfuric acid as
Streptomycetes, Actinomadura, Dermatophilus, decolorizing agent)
Nocardiopsis, and the Thermophilic → Histopathologic examination
Actinomycetes o Gomori’s methenamine-silver (GMS)
◊ Assoc. with chronic,granulomatous lesions of skin → Biopsy and drainage material: look for granulles
(mycetomas) → For granules:
o Washed in saline
Mycetomas: infx of sibQ tissues = tissue swelling and o Emulsified in 10% KOH
drainage of sinus tracts o Crushed in bet 2 slides
◊ Traumatic inoculation: lower limbs, caused by o Gram stained
fungi Molecular Methods
◊ Actinomycetoma: mycetoma caused by
actinomycete Amplification techniqies

Nonmycetomic infection: occur in px infected with HIV
Thermophilic actinomycetes: hypersentivity
pneumonitis
 Allergic rxn to agents
 Occupational dse of farmers, factory
 workers
PCR withn 16S Rrna sequence: examine relatedness
among genera within non-acid-fast aerobic
actinomycetes and thermophilic actinomycetes
MicroSeq System: identification
PCR paired with restriction endonuclease analysis:
identify commonly isolated Nocardia spp.
DNA sequencing for genes
֍ 16S r RNA
֍ Heat shock protein gene
֍ Housekeeping gene = secA1
o Bacterial transport molecule
PCR of 16S r RNA with choE gene= identification of
Rhodococcus
*not for clinical laboratory but for taxonomy,
epidemiologic, and research studies*
Cultivation
 Do not have complex grow reqs
 Grow on routine lab media:
o S-BA
o Chocolate
o SAabouraud dextrose (SDA)
o Löwenstein-Jensen
o BHI
 Aerobic actinomyces: grows slow and may be
overgrown by other bacteria in contaminated
specimens
o Nocardiae: visible growth requires 48-72
hours of incubation
 Solid medium: paraffin (sole source of carbon)
o Effective for isolation Nocardia spp
o Rapidly growing mycobacteria
 BCYE: selective for Legionella spp
o With polymixin, anisomycin, and either
vancomycin/cefamandole
o Effective for nocardiae recovery
 MTM and CNA
 Sabouraud dextrose agar (SDA): Nocardia spp
grows and fungal media containing
cycloheximide (e.g. Mycosel agar)
o Nocardia can withstand decontamination
procedures for mycobacteria
 BHI with chloramphenicol and cycloheximide:
selective medium
o Enhance isolation
 Grow on 35°C, INCREASED on 30°C
o Agars should be heated on both temp (4
agar plates)
o 2-3 weeks of incubation
o Vary bet. 1-19 days
 Suspected aerobic actinomycetes: incubated for
minimum of 3wks
 Dx of themophilic actinomycetes: grow rapidly on
TSA with 1% yeast extract
o Grow on 50°C or greater (characteristic)
Approach to Identification
1) Acid-fast: performed first to rule out rapidly
growing mycobacteria
2) Modified acid-fast stain
(+): Nocardiam Rhodococcus,
Tsukamurella/Gordonia
(-): variability of organisms
Groupings of aerobic actinomycetes
 Gram-stain morphology
 Modified acid-fast stain
 Presence/absence of aerial hyphae + spores,
number and arrangement
 G/NG in nutrient broth with lysozyme
(250µg/mL)
 Other tests:
o Urea hydrolysis
o Nitrate reduction
o Ability to grow anaerobically
Identification of Nocardia: time consuming (2 weeks)
Phenotypic tests
 Use of casein, xanthine, and tyrosine hydrolysis
 Growth at 45°C
 Opacification of Middle-brook agar
 AST patterns
*referred to a RL*
Antimicrobial Susceptibility Testing and Therapy
For Nocardia spp
 Broth microdilution with cation-supplemented
Mueller-Hinton broth
 Modified disk diffusion
 Agar dilution
 E-test
 Radiometric growth index
◊ If needed, send isolates to RL
For other actinomycetes
» No standardized methods
» AST for Rhodococcus & Gordonia spp: guide for
directing therapy
*no effective antimicrobial therapy available for
hypersensitivity pneumonitis caused by the
thermophilic actinomycetes*
No vaccine
Section 14: Chapter 42 General Characteristics
Mycobacteria Complex- 2 or more spp. which cannot be
distinguished easily or has little or no medical
Introduction importance
 Aerobic Includes: CAPABLE OF CAUSING TB
o May grow in reduced O2  M. tuberculosis
 Non-spore forming (except M.marinum)  M. bovis
 M. bovis BCG
 Nonmotile
 M. africanum
 Very thin
 M. caprae
 Slightly curved/straight rods  M. microti
o 0.2-0.6 X 1-10 µm  M. canettii
 Some may show branching  M. mungi
 Mycobacterim = genus Mycobacteriaceae  M. orygis
 Related to Mycobacterium: Nocardia,  M. pinnipedii
Rhodococcus, Tseukamurella, and Gordonia → Slow growers and produce nonpigmented
colonies
Mycobacterium spp:
→ Cell wall: N-glycolmuramic acid Epidemiology and Pathogenesis
o Instead of N-acetlymuramic acid Epidemiology
o Has high lipid content M. tuberculosis complex exhibit genetic homogeneity
o Hydrophobic permeability barrier M.tuberculosis- most common pathogen to cause
→ Difficult to stain using basic aniline dyes human tuberculosis
→ Considered as gram positive  M.tuberculosis complex should be isolated to
→ Resists decolorization with acidified alchohol (3% growth in tissues of humans and other warm-
hydrochloric acid) in prolonged application of blooded animals
basic fuchsin dye or heating of dye Tuberculosis common found among
→ Acid fastness: important property based from its a) Poor
cell wall b) The homeless
→ Rapid-growing mycobacteria (RGM): grows in c) IV drug users
less than 7 days in LJ (Lowenstein-Jensen) d) Alcoholics
medium e) The elderly
→ Many spp. are slow growing due to its cell surface f) Medically underserved pop.
o Hydrophobicity: constitutes to its clumping
 Nutrients does not easily pass Pathogenesis
through  Inhalation can lead to infection
→ Generation time: time required for a cell to divide  15-20% of infected person are likely to develop
into two independent cell the disease
o 20hours to 36 hours for M.ulcerans  M.bovis: Ingestion of milk from infected cows
→ Slow-growing mycobacteria: require >7 days to o can penetrate GI mucosa and invade
produce colonies on solid media lymphatic tissue of the oropharynx
o Visible colonies in 2-60 days at optimum o M. bovis, bacillus Calmette-Guerin
temp. (BCG) – used in immunization
→ Nontuberculous environmental mycobacteria  M. africanum – manifest characteristic
(NTM) intermediately between M.tuberculosis and
→ Prominent pathogens: Mycobacterium M.bovis
tuberculosis complex, Mycobacterium leprae, and o Causes half of the cases in West Africa
Mycobacterium ulcerans o Or px who recently resided in Africa
→ 2 major groups based on epidemiology and  M.caprae – identified by its susceptibility to
association with disease: M.tuberculosis complex pyrazinamide
and NTM. o Contributes to 31% cases of human
tuberculosis
o Reservoir hosts: goats, cattle, sheep, pigs,
Mycobacterium tuberculosis Complex wild boars, deer, and fox
Tuberculosis – also called as “consumption”  M.microti – rodent, guinea pigs, rabbits, cats,
llamas, and meerkats
 o Fails to grow in culture
o TB in both immunocompetent and
immunosuppressed
 M. canetti – cases of lymphadenitis and
generalized TB in immunocompromised
individuals
o Assoc. with infection in px who recently
resided in Africa
 M.pinnipedii – from sea lions to humans
o Granulomatous lesions in lymph nodes,
lungs, pleura, and spleen
 Reservoir hosts
o Banded mongoose (M.mungi)
o Large animals: gazelles, antelopes,
waterbucks, and oryxes (M.orygis)
Spectrum of Disease
 Tb mimics pneumonia, neoplasm, or fungal
infections
 Px infected of complex can be ASYMPTOMATIC.
ACUTELY SYMPTOMATIC
Symptomatic: systemic symptoms
 Pulmonary SSX
 Other organ involvement (kidneys)
 Combination
Primary TB: dse of the respiratory tract
SSX:
a) Low-grade fever
b) Night sweats
c) Fatigue
d) Anorexia
e) Weight loss
Pulmonary TB: productive cough, low-grade fever,
chills, myalgias (aches), sweating
- SSX sim. to pneumonia, influenza, and acute
bronchitis
Granulomas: hard tubercle formed in the lung from
the lymphocytes, macrophages, and cellular
pathology
- Giant cell formation
Mycobaterium antigen conc = hypersensitity =
tissue necrosis
*no granuloma forms – solid/semisolid caseous
material is left on primary lesion site
 Primary active TB: spread via lymph system or
hematogenously
o Meningeal/military (disseminated TB)
 Pc with depressed cellular
immunity
Organs affected by M. tuberculosis complex:
 Genitourinary tract
 Lymph nodes (cervical Lymphadenitis)
 CNS (meningitis)
 Bone and joint (arthritis and osteomyelitis)
 Peritoneum
 Pericardium
 Larynx
 Pleural lining (pleuritis)
Tuberculin skin test/ Purified Protein derivative – for
diagnosing disseminated TB
 Based on delayed hypersensitivity cell-
mediated immunity of px to certsian
antigenic component of the bacteria
Latent TB: no ssx
- Not infectious and does not have active TB
- Organism present in granulomas
- May progress to active dse = reactivation
tuberculosis
o Occurs when cellular immunity is
suppressed or damaged due to
change in lifestyle
 HIV pxt are susceptible to developing active TB
 Anergenic: lack biogenic response to tuberculin
test
o Primary indicator of px infected with
M.tuberculosis
 To determine if person is infected with
M.tuberculosis
o Culture extract is injected intracutaneously
o 48-72 hours: infected shows
hypersensitivity rxn
 Eythema (redness)
 Firmness as a result of influx of
immune cells
 Diameter of induration
 T-Spot TB – next-day results and does not
require a follow-up visit with a physician
o Measures T cells that have bene activated
by M.tuberculosis antigens
 ELISA: QuantiFERON-TBA Gold: measures the
component of cell-mediated immune response to
M. tuberculosis to diagnose later TB infx. and TD
dse.
Nontuberculous Mycobacteria
 Opportunistic pathogens
 MOT: trauma, inhalation of aerosols, and
ingestion
 Can be nosocomial or iatrogenic infection
Runyoun groups I to IV
Slow-Growing Nontuberculous Mycobacteria
Mycobaterium spp: synthesize carotenoids (group of
yellow to red pigments)
1) Photochromogens: produce colonies that require
light to form pigments
2) Scotochromogens: produce pigmented colonies
in either in the dark or light
3) Nonphotchromogens: produce unpigmented
colonies in either dark or light
a. M. terrae complex (terrae, trivale, and
nonchromogenicum)
b. M. gastri
i. Nonpathogenic
c. M. avium complex (MAC): able to cause
infection to human host
Mycobacteria avium Complex
 Highly active antiretroviral therapy (HAART):
reduced infx. in px with AIDS
o M. avium
o M. intreacellulare
o M. avium subsp. Aviium
o M. avium subsp. Paratuberculosis
o M. avium subsp. Silvaticum (wood pigeon
bacillus)
o M. avium subsp. Hominissuis
o M. arosiense
o M. vulneris
o M. marseillense
o M. bouchedurhonense
o M. chimera
o M. colombiense
o M. timonense
Epidemiology and Pathogenesis
MAC: affect both immunocompromised and
immunocompetent
- Assoc in infx in px with AIDS
- Isolated from natural water, soil, dairy
products, pigs, chicken, cats, and dogs
- Acquired by inhalation or ingestion
- Assoc with lymphadenitis in children and
disseminated infx in px with HIV
Cultures: opaque, glossy, white, colony morphology
or produce smaller translucent colony morphology
- Transparent colonies are more virulent = durg
resistant
M. avium subsp. Paratuberculosis: known to cause
inflammatory bowel disease (John Disease)
☼ isolated also in px with Crohn’s Dse
☼ extremely fastidious: require mycobactin
o produced by M.phlei – saprophytic
strain
o 6-18 months for primary isolation
Rapid Growing Nontuberculous Mycobacteria
- Grows on solid media in 7 days or earlier
o M. abscessus subsp. Abscessus
o M. chelonae
o M. fortuitum
- Can grow on routine media
- Weakly gram positive rods resembling
diptheroids
- Found in soil, marshes, rivers, tap water, and
marine and terrestrial life forms
- HCAInfx: BM transplantations, wound infx,
and catheter sepsis
- MOE: skin and subQ due to trauma,
injections, surgery, or animal contact
- Can cause disseminated cutaneous
infections.
- NTM: M. fortuitum, M.chelonae, M. abscessus
subsp. Abscessus
- Assoc with posttraumatic wound infection
Noncultivatable Nontuberculous Mycobacteria –
Mycobacterium leprae
 Cause leprosy (Hansen disease)
Leprosy: chronic disease of the skin, mucous
membranes, and nerve tissue
 Cultivated in armadillo and footpads of mice
 Hypopigmented skin lesions and peripheral nerve
involvement – skin smear (+) for acid-fast bacilli
 1°Reservoir: infected humans
 MOT: inhalation or contact with infected skin
o Inhalation of discharge in the
nasalsecretons of the infected
 Silent phase: multiplication of leprosy bacilli in the
skin in macrophages
 Intermediate phase: multiplication in peripheral
nerves causing sensory impairment
Tuberculoid leprosy: localized form
- Do not have immune defect
- Few organisms
- Localized to skin and nerves
Lepromatous leprosy: disseminated form
- Anergic to M.leprae
- Defect in cell-mediated immunity
- Numerous acid-fast bacilli
Laboratory Diagnosis of Mycobacterial Infections
◊ BSL 2 practices
◊ BSC II safety precautions
◊ Disinfectants: Amphyl, 0.05%-0.5% sodium
hypochlorite, or phenol-soap mixtures
Specimen Collection and Transport
Specimens
→ Sputum
→ Tracheal or bronchial aspirates
→ Spx in bronchial alveolar lavage
→ Urine
→ Gastric aspirates
→ Tissue (biopsy)
→ CSP
→ Pleural and pericardial fluid
→ For immunocompromised px: blood or fecal spx
Specimen of Choice: Spontaneously produced Fuchsin Acid-Fast
sputum Ziehl-Neelsen: heating the slide, hot stain procedure
For gastric lavage spx, SOC: for children <3 year olds Kinyoun: cold stain procedure
Urine: 3 morning glass, clean-catch midstream urine
CSF: 10Ml Antigen-Protein Detection
Other fluids: 10-15 Ml (pleural, peritoneal, and ELISA: lipoarabinomannan (LAM) – component of
pericardial fluids – with Luer tip cap) cell wall of M.tuberculosis
- 2 cultures:
o 30° - M. haemophilum, M. marinum, Immunodiagnostic Testing
o M.ulcerans T-SPOT-TB: requires peripheral blood mononuclear
Blood: SPS, heparin, citrate cells
Wounds, Skin lesions, and Aspirates: QFNG-IT: stimulation of T-cell interferon-gamma in
- Aspirate: best for culturing skin lesion/wound whole blood
- Amier’s or Stuart’s medium: transport medium
Cultivation
Contaminated Specimens Solid Media
 Rapidly growing mycobacteria: susceptible to 1) Serum (albumin) agar: Middlebroook 7H10
high/prolonged exposure to 2%/more sodium 2) Egg-potato base medium: Lowenstein-
hydroxide (NaOH) Jensen/L-J
 Incubated at 35°C in the dark with 5%-10% CO2
Specimen Rejection and h ugh humidity
1) Insufficient volume CAP: with X-factor (hemin), 25-33°C for M.
2) Contamination with saliva haemophilum
3) Dried swabs RGM: 28-30°C
4) Pooled sputum or urine M. marinum, M. ulcerans: 25-30°C
5) Leaking or broken container
6) Prolonged specimen collection and  Isolates grow between 3-6 weaks
processing
֍ NaOH, oxalic acid, and mild HCl: reduces
recovery for M.ulcerans Conventional Phenotypic Test
֍ Liquefying agents: NALC, diothiothreitol Preliminary identification:
(Sputolysin), and enzymes  Rate of growth
 Colonial morphology
Special Considerations  Colonial texture
 Aerosol-induced sputum is treated as sputum  Pigmentation
 Gastric lavage: processed within 4 hours with  Permissive incubation
10% sodium carbonate Growth rate:
 Urine: Sputolysin-oxalic acid method - Rapid growers: 3-4 days, 7 days
 Fecal spx: decontaminated with oxalic acid or - False rapid growth: M. flavescens
NALC-NaOH method Pigment production:
 Swab/wounds: Middlebrook 7H9  M. kansasii: yellow after few hours of light
 Tissue: sterile saline (85%) or sterile 0.2% bovine exposure
albumin  M. szulgai: sctochromogen (35°C),
Nonpipgmented when grown at 25°C
Direct Detection Methods
Acid-Fast stains Biochemical Testing
Mycolic acid: contribute to acid-fastness Niacin: important in the oxidation-reduction reactions
- Long-chain, multiple cross-liked fatty acids of mycobacterial metabolism
 Mycobacteria in gram staining: “gram neutral” or - (+): preliminary evidence, buffered colored,
“gram ghosts” slow-growing, rough colony = may be M.
 Rapid growers: acid-fast variable tuberculosis
Fluorochrome Stain: recommended for laboratoues
that have UV microscope Nitrate Reduction: identifying M. tuberculosis, M.
- More sensitive kansasii, M. szulgai, and M. fortuitum
- Bacilli against a dark background
 - Ability of acid-fats bacilli to reduce nitrate the
is influenced by age of colonies, temperature,
pH, and enzyme inhibitors
Catalase: M.gastri – produce intracellular enzyme
catalase which splits H2O2 into water and oxygen
- Semiquantitative catalase test: activity of the
enzyme as determined by the height of a
column of bubbles of oxygen
- Heat-stable catalase test: ability of the
catalase enzyme to remain active after
heating
o 68°C for 20 minutes
o M. tuberculosis, M. bovis, M. gastri,
and M. haemophilum: inactivated
Tween 80 Hydrolysis: used for differentiating
photochromogens, nonchromogens, and
scotochromogens
Tellurite Reduction: ability to reduce tellurite in 3-4
days distinguishes MAC from other nonchromogenic
spp.
Arylsulfatase: present in most mycobacteria
- Varied to distinguish the different forms of the
enzyme
- The rate at which enzyme breaks down
phenophthalein disulfate into phenolphthalein
o Red color in the presence of sodium
bicarbonate
Growth Inhibition by Thiophene-2-Carboxylic Acid
Hyrazide (TCH)
- Growth inhibition is used to distinguish
M.bovis from M.tuberculosis
o M.bovis: unable to grow in the
presence of 10 mg/ml of TCH
Antimicrobial Susceptibility Testing and Therapy
MDR tuberculosis
 Rifampin (R)
 Isoniazid (R)
Extensively drug-resistant tuberculosis (XDR TB)
 Rifampin (R)
 Isoniazid (R)
 Quinolones (R)
 Aminoglycosides (R)
 Capreomycin (R)
M. tuberculosis Complex
 Isolates must be saved in 10% skim milk inn
distilled water
 -70°C for future additional studies
Indirect Susceptibility Testing: uses a culture as the
inoculum source
Conventional Methods
Critical concentration of a drug: the amount of drug
required to prevent growth above the 1% threshold of
the test population of tubercle bacilli
Primary drugs: the five antimicrobials tested for the
initial isolates of M. tuberculosis
Therapy
→ Directed against M. tuberculosis depends on
the susceptibility of the isolate
o Isoniazid, rifampin, ethambutol, and
pyrazinamide
o Streptomycin
→ The preferred therapy for initial treatment
o Isoniazid and rifampin for additional 18
weeks
→ Two-drug regimen is isoniazid (INH
isonicotinoylhydrazine) and rifampin
o Administered for 9 months
o If with pyrazinamide in the first 2
months, drug administration is reduced
to 6months
Nontuberculous Mycobacteria
Pulmonary infection for MAC
☼ Clarithromycin
☼ Rifampin
☼ Ethambutol (streptomycin/amikacin: severe
disease)
Pulmonary infection for M.kansasii
֍ Isoniazid
֍ Rifampin
֍ Ethambutol
Skin and soft tissue infection with M.marinum
 Clarithromycin & ethambutol
 Clarithromycin and rifampin
 Rifampin and ethambutol
Pulmonary infections with M. abscessus
→ Multidrug regimen
o Clarithromycin
Prevention
Prophylactic chemotherapy with INH
BCG vaccine – the only vaccine against tuberculosis